Organelle Cross Talk and Membrane Dynamics

Lead Research Organisation: University College London

Abstract

Our cells are divided into distinct membrane-bound compartments some of which form pathways that transport materials into and out of cells. These membrane trafficking pathways control key cellular processes including nutrient uptake, clearance of activated growth factor receptors, and the secretion of hormones and neurotransmitters. However our understanding of membrane trafficking systems remains incomplete. Our research integrates cell biology, biochemistry, and system-wide approaches to better understand how membrane trafficking and cell signalling pathways allow cells to respond to cues in their environment, such as growth factors, nutrients, and stress. Our long-term goal is to develop new therapeutics for the treatment of diseases caused by membrane trafficking defects.

Technical Summary

Cells and tissues communicate with one another through the release of hormones and neurotransmitters that are recognized by signaling receptors on target cells. As such, cell-cell communication is tightly coupled to membrane trafficking pathways that control the secretion of signaling factors and the targeting of cell-surface receptors. Our group is investigating the structural requirements and physiological roles for membrane lipid dynamics in cell signaling and membrane trafficking pathways. This work is revealing novel roles for ‘intracellular synapses’ between the between the endoplasmic reticulum (ER) and plasma membrane (PM). We are elucidating how the assembly and disassembly of ER-PM contacts are regulated and how distinct ER-PM contacts modulate phosphoinositide (PI) kinase regulatory networks. PI kinase signalling networks control several key cellular processes including cell growth and survival, cell polarity, and membrane trafficking pathways. Consequently, defects in PI kinase regulatory networks have been implicated in numerous human diseases including cancer, diabetes, and neurodegenerative disorders. It is vital that we understand how cells maintain and use these essential signalling molecules. We are currently undertaking multidisciplinary approaches–including cell biological assays, high-resolution microscopy, proteomics, lipidomics, and biochemical approaches–to make new discoveries into the regulation of PI kinase signaling networks by ER-PM cross talk. We expect that these investigations will reveal new insights into the complex regulatory processes that ensure the temporal and spatial specificity of membrane trafficking systems, including regulated exocytosis and endocytosis necessary for pulsatile insulin secretion and neurotransmission.
Moreover, we have found that ER-PM cross talk is essential for normal ER morphology and function. Loss of ER-PM contacts causes dramatic changes in ER organization and architecture, resulting in activation of ER stress response pathways. Recent findings have placed ER stress as a key component of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS). We are currently elucidating the vital roles for PM-ER contacts in ER membrane homeostasis, as well as the essential cellular pathways induced in response to membrane stress.

Publications

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Malia PC (2018) Control of vacuole membrane homeostasis by a resident PI-3,5-kinase inhibitor. in Proceedings of the National Academy of Sciences of the United States of America

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Nishimura T (2020) Specialized ER membrane domains for lipid metabolism and transport. in Biochimica et biophysica acta. Molecular and cell biology of lipids

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Shin JJH (2020) pH Biosensing by PI4P Regulates Cargo Sorting at the TGN. in Developmental cell

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Stefan CJ (2018) Building ER-PM contacts: keeping calm and ready on alarm. in Current opinion in cell biology

 
Description The Osamu Hayaishi Scholarship for Study Abroad (The Japanese Biochemical Society)
Amount ¥4,838,823 (JPY)
Organisation Japanese Biochemical Society 
Sector Learned Society
Country Japan
Start 01/2018 
End 12/2018
 
Title High content screens for modulators of motor neuron disease 
Description Amyotrophic Lateral Sclerosis (ALS) is inexorably fatal because no treatment halting or preventing the disease is available. Mechanisms implicated in the disease process are poorly defined and highly complex. To identify chemical entities that target the underlying toxicity, it is essential to embrace unbiased and innovative approaches in which the search for effective therapeutics is accompanied and supported by an increased understanding of disease mechanisms. Dr. Stefan is a scientist with a longstanding interest in the biology of VAPB, the ALS8 causative gene. Dr. Stefan and his coworkers have conducted an HCS in a neuronal cellular model of ALS8 recapitulating major hallmarks of the human disease. Because inclusion formation is an easy assayable phenotype and represents a fundamental pathological feature, a screen of small molecule compound libraries (with more than 45,000 compounds currently available) was conducted to identify compounds that disrupt inclusion formation. Subsequently, compounds were tested in a series of phenotypic and functional readouts including changes in neurite extension, quantitative evaluation of ER stress, alterations in lipid metabolism, intracellular translocation of the disease protein and neuronal cell survival. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact Candidate compounds have been identified and further tested in a series of phenotypic and functional readouts including changes in neurite extension, quantitative evaluation of ER stress, alterations in lipid metabolism, intracellular translocation of the disease protein and neuronal cell survival. Candidate compounds are now in tests to validate their effectiveness in other forms of ALS. 
 
Title High throughput assays for regulators of PI kinases and phosphatases 
Description High throughput assays for small molecule regulators of PI kinases and phosphatases 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Phosphoinositide (PI) kinase signalling networks control several key cellular processes including cell growth and survival, cell proliferation and differentiation, cell polarity and migration, and membrane trafficking pathways. Defects in PI kinase regulatory networks have been implicated in numerous human diseases including cancer, diabetes, and neurodegenerative disorders. Our assays are designed to (1) validate PI kinases and phosphatases as potential drug targets, and (2) to identify small molecule compounds (chemical) that directly target and regulate these enzymes. We expect these studies will be particularly relevant to neuronal physiology, as defects in PI metabolism have been implicated in several neurological and neurodegenerative disorders including Down's syndrome, Alzheimer's disease, Parkinson's disease, bipolar disorders, and autism. 
 
Title YPA-Yeast Protein Atlas 
Description At UCL, the Stefan lab is undertaking system-wide approaches to make new discoveries in the cell biology. As part of this research program, Dr. Stefan's group is carrying out high-throughput genomic screens using yeast cells as a model system. Yeast cells are widely used in system-wide genomic studies, such as yeast genetic approaches (YGA), epistatic mini-array profiling (E-MAP), and chemical-genetic profiling techniques. To apply functional genomics approaches to cell biological problems, this new lab will combine existing yeast genomic approaches (YGA) with high-content quantitative imaging for system-wide analysis of changes in protein localization and abundance (YPA) under a variety of genetic, environmental, and chemical stresses using rapid image acquisition and quantitative analysis. Combined YGA-YPA approaches will provide new ways to monitor dynamic changes in cellular architecture. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact With the establishment of a new high-throughput yeast cell biology facility at the LMCB, UCL will become one of only a few research institutions in the world capable of coupling functional genomics with high-content protein localization screens in yeast. The yeast protein atlas (YPA) database and technology will increase the resolution of existing yeast genetic approaches (YGA), enabling the detailed investigation of numerous biological processes. As such, this new YPA database and technology will benefit many yeast cell biology groups across the UK. 
 
Description Control of lysosome membrane homeostasis 
Organisation University of Osnabrück
Department School of Biology/Chemistry Osnabrück
Country Germany 
Sector Academic/University 
PI Contribution Lysosomes have an important role in cellular protein and organelle quality control, metabolism and signaling. On the surface of lysosomes, the PIKfyve/Fab1 complex generates phosphatidylinositol 3,5-bisphosphate, or PI(3,5)P2, which is critical for lysosomal membrane homeostasis and for lysosomal signaling during acute cellular stress. However, it is still unknown how he PIKfyve/Fab1 complex senses changes in the lysosomal membrane and is regulated during cellular stress. We are elucidating regulation of the PIKfyve/Fab1 complex with a direct influence on PI(3,5)P2 levels and vacuole homeostasis.
Collaborator Contribution The Ungermann lab studies lysosomal membrane dynamics and cross talk with PIKfyve/Fab1-mediated PI(3,5)P2 synthesis to counterbalance membrane stress.
Impact Malia P, Numrich J, Nishimura T, Gonzalez Montoro A, Stefan C, and C Ungermann (2018). Control of vacuole membrane homeostasis by a resident PI3P 5-kinase inhibitor. Proc Nat Acad Sci. 115(18):4684-4689. PMID: 29674454 PMCID: PMC5939101 DOI: 10.1073/pnas.1722517115
Start Year 2017
 
Description Molecular Dynamics and Modelling Membrane Organization at the Nanoscale Level 
Organisation Max Planck Society
Department Max Planck Institute of Biophysics
Country Germany 
Sector Charity/Non Profit 
PI Contribution The Stefan lab is investigating vital roles for membrane lipid dynamics during organelle biogenesis and homeostasis. Using biophysical and theoretical approaches, we have elucidated essential roles for conserved lipid transfer proteins that establish the precise mechano-chemical and biophysical membrane properties necessary for plasma membrane organization.
Collaborator Contribution The Hummer group at the Max Planck Institute of Biophysics uses molecular dynamics simulations to reveal lipid organization in membrane bilayers at the molecular level.
Impact Nishimura T, Gecht M, Covino R, Hummer G, Surma M, Klose C, Arai H, Kono N, and CJ Stefan (2019). Osh Proteins Control Nanoscale Lipid Organization Necessary for PI(4,5)P2 Synthesis. Molecular Cell. 75: 1043-1057. PMID: 31402097 PMCID: PMC6739424 DOI: 10.1016/j.molcel.2019.06.037 This is a multi-disciplinary collaboration involving cell biological, biochemical, biophysical, and computational (modeling) approaches.
Start Year 2018
 
Description Quantitative Lipid Mass Spectrometry 
Organisation University of Tokyo
Country Japan 
Sector Academic/University 
PI Contribution The Stefan lab is investigating vital roles for membrane lipid dynamics during organelle biogenesis and homeostasis. Using lipidomics approaches, we are elucidating the mechanisms that establish and maintain the unique membrane lipid composition of distinct organelles (Nishimura et al in revision).
Collaborator Contribution Prof. Nozomu Kono's group at the University of Tokyo uses performs quantitative lipid mass spectrometry in parallel with the Stefan laboratory.
Impact Nishimura T, Gecht M, Covino R, Hummer G, Surma M, Klose C, Arai H, Kono N, and CJ Stefan (2019). Osh Proteins Control Nanoscale Lipid Organization Necessary for PI(4,5)P2 Synthesis. Molecular Cell. 75: 1043-1057. PMID: 31402097 PMCID: PMC6739424 DOI: 10.1016/j.molcel.2019.06.037 This is a multi-disciplinary collaboration involving cell biological, biochemical, biophysical, and computational (modeling) approaches.
Start Year 2018
 
Description Role of Calcium and Membrane Lipid Dynamics in Immune Cell Responses 
Organisation Cornell University
Department Department of Chemistry and Chemical Biology
Country United States 
Sector Academic/University 
PI Contribution Ca(2+) mobilization in response to cross-linking of IgE bound to its high affinity receptor, FceRI, on mast cells is central to immune allergic responses. Stimulated tyrosine phosphorylation caused by this cross-linking activates store-operated Ca(2+)entry that results in sustained Ca(2+)oscillations dependent on Rho family GTPases and phosphoinositide synthesis. Coupling of the endoplasmic reticulum (ER) Ca(2+)sensor, stromal interaction molecule 1 (STIM1), to the Ca(2+)-selective channel, Orai1, is regulated by these elements and depends on membrane organization, both at the plasma membrane and at the ER. Mitochondria also contribute to the regulation of Ca(2+)mobilization, and we describe recent evidence that the ER membrane protein vesicle-associated membrane protein-associated protein (VAP) plays a significant role in the coupling between ER and mitochondria in this process. In addition to granule exocytosis, Ca(2+)mobilization in these cells also contributes to stimulated outward trafficking of recycling endosomes and to antigen-stimulated chemotaxis, and it is pathologically regulated by protozoan parasitic invasion.
Collaborator Contribution This work is discovering regulatory mechanisms for mast cell responses by calcium and phosphoinositide signaling networks.
Impact To date, one publication has resulted from this collaboration. Roles for Ca2+ mobilization and its regulation in mast cell functions: recent progress David Holowka, Marcus Wilkes, Christopher Stefan, Barbara Baird Biochemical Society Transactions Apr 11, 2016, 44 (2) 505-509; DOI: 10.1042/BST20150273
Start Year 2014
 
Description Targeting Membrane Homestasis in Motor Neuron Disease 
Organisation University of Edinburgh
Department Euan Macdonald Centre for Motor Neurone Disease Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Amyotrophic Lateral Sclerosis (ALS) is inexorably fatal because no treatment halting or preventing the disease is available. Mechanisms implicated in the disease process are poorly defined and highly complex. To identify chemical entities that target the underlying toxicity, it is essential to embrace unbiased and innovative approaches in which the search for effective therapeutics is accompanied and supported by an increased understanding of disease mechanisms. Drosophila is emerging as a promising animal model for testing therapeutic options mainly because of excellent in vivo readouts of pathology, numerous genetic resources available and high degree of conservation of genetic pathways between flies and humans. Mammalian models have the advantage of being much more similar to humans but the length of the time and the cost required to perform experiments comparable to those possible in flies, can be prohibitive. Cell-based models can be used for high-throughput drug screens but they may not recapitulate the response of the entire organism. Dr. Stefan is a scientist with a longstanding interest in the biology of VAPB, the ALS8 causative gene. Dr. Stefan and his coworkers have conducted an high-content screen (HCS) in a neuronal cellular model of ALS8 recapitulating major hallmarks of the human disease. Because inclusion formation is an easy assayable phenotype and represents a fundamental pathological feature, a screen of small molecule compound libraries was conducted to identify compounds that disrupt inclusion formation. Subsequently, compounds were tested in a series of phenotypic and functional readouts including changes in neurite extension, quantitative evaluation of ER stress, alterations in lipid metabolism, intracellular translocation of the disease protein and neuronal cell survival.
Collaborator Contribution Dr. Pennetta's lab generated a Drosophila model of ALS8 in which the expression of the pathogenic transgene in the eye photoreceptors or in all neurons, induces neurotoxicity that can be monitored by measuring the reduction in eye size, progressive motor abnormalities and early organismal death. These phenotypic readouts are easy-to-score and can be used for quantitative and sensitive analyses of compound-mediated effects. The phenotypic analysis could also be extended to include aggregate formation, alterations in synaptic structure and function and muscle defects. A genome-wide screen aimed at identifying genetic modifiers of disease phenotypes in a fly model for ALS8 identified genes and pathways important for ALS pathogenesis, indicating potential targets for therapeutic intervention. Moreover, these pathways largely overlap with those identified by a high-content screen (HCS) of small molecule compounds performed in an ALS8 cell-based model by the lab of Dr. C. Stefan at University College London, UK. In a collaborative effort with Dr. Stefan's lab, a multisystem, cross-species approach in which Drosophila is be used for: 1) exploring the possibility of targeting the Hippo pathway for therapeutic intervention; 2) testing and mechanistically validate compounds identified by the cell-based screen with particular emphasis on those affecting proteins and pathways shown to be important for ALS pathogenesis by the genetic modifier screen.
Impact In progress
Start Year 2017
 
Description The Architecture of Organelle Contacts at the Ultrastructural Level 
Organisation Max Planck Society
Department Max Planck Institute of Biochemistry
Country Germany 
Sector Academic/University 
PI Contribution Membrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) play fundamental roles in all eukaryotic cells. The Stefan laboratory is investigating the molecular architecture and regulation of junctions between the endoplasmic reticulum and the plasma membrane (ER-PM), in their cellular context and in vitro. ER-PM junctions are conserved intra-cellular synapses that control inter-organelle calcium and membrane lipid dynamics during regulated exocytosis and neurotransmission. Our previous work has revealed the molecular machinery that forms ER-PM junctions and a role for ER-PM junctions in modulating lipid metabolism and calcium signaling (Omnus et al, MBoC 2016; Manford et al, Dev Cell 2012; Stefan et al, Cell 2011). However, the individual contributions of the ER-PM tethering molecules and their regulation during triggered exocytosis are poorly understood. Electron microscopy (EM) approaches are critical to understand how the exact spacing of ER-PM junctions is determined and how the organization of ER-PM contacts defines their function. In collaboration with the EM core facility at the MRC Laboratory for Molecular Cell Biology and the Max Planck Institute for Biochemistry at Martinsried, we are currently employing cryo-electron tomography (cryo-ET) approaches, using vitrified cells and keeping cellular components in a frozen-hydrated state, to best permit the study of ER-PM contacts in their native cellular context. These investigations will provide unparalleled structural information into the regulation of ER-PM cross talk by calcium and membrane lipid dynamics.
Collaborator Contribution The Busnadiego-Fernandez lab at the Max Planck Institute for Biochemistry provides expertise in cryo-electron tomography to study key determinants of cell architecture and morphology.
Impact Collado D, Kalemanov M, Martinez A, Bourgoint C, Thomas F, Loewith R, Martinez-Sanchez A, Baumeister W, Stefan CJ, and R Busnadiego-Fernandez (2019). Tricalbins Control ER Curvature to Maintain PM Integrity. Dev Cell, 51 (4), 476-487.e7 PMID: 31743662 PMCID: PMC6863395 DOI: 10.1016/j.devcel.2019.10.018 This is a multi-disciplinary collaboration involving cell biological, biophysical, and computational (modeling) approaches.
Start Year 2017
 
Description pH Biosensing and PI Kinase Signaling 
Organisation University of British Columbia
Country Canada 
Sector Academic/University 
PI Contribution Phosphoinositides, diacylglycerolpyrophosphate, ceramide-1-phosphate, and phosphatidic acid belong to a unique class of membrane signaling lipids that contain phosphomonoesters in their headgroups having pKa values in the physiological range. The phosphomonoester headgroup of phosphatidic acid enables this lipid to act as a pH biosensor as changes in its protonation state with intracellular pH regulate binding to effector proteins. Here, we demonstrate that binding of pleckstrin homology (PH) domains to phosphatidylinositol 4-phosphate (PI4P) is dependent on intracellular pH, indicating PI4P is a pH biosensor. pH biosensing by PI4P in response to nutrient availability and cellular status governs protein sorting in the secretory and endocytic pathways. Thus, pH biosensing by TGN PI4P allows for direct metabolic regulation of protein trafficking and cell growth.
Collaborator Contribution The Loewen laboratory has measured effects of pH on phosphoinositide properties.
Impact Shin J, Liu P, Chan L, Ullah A, Pan J, Borchers C, Burke C, Stefan C, Smits C and CJR Loewen (2020). pH Biosensing by PI4P Regulates Cargo Sorting at the TGN, Developmental Cell, 52 (4), 461-476.e4 PMID: 31928972 DOI: 10.1016/j.devcel.2019.12.010
Start Year 2018
 
Description Interview for national television program 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Appeared on TV broadcast of "How to lose weight well" as cell membrane expert and provided a lab demonstration.
Year(s) Of Engagement Activity 2018